The neuron has a remarkable ability to release quanta of neurotransmitters in less than a millisecond in response to the pulse of Ca2+. This fast synchronized release constitutes the fundamental basis of major brain activities. The release requires vesicle fusion, which is orchestrated by the fusion machine SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, a priming agent Complexin (Cpx), and the Ca2+ switch Synatotagmin 1 (Syt1). This research project uses the innovative single-vesicle fusion assay to investigate the mechanism by which SNARE-dependent vesicle fusion is regulated by Cpx, Syt1, and Ca2+. The single-vesicle fusion assay makes it possible to dissect the sequential intermediate stages of vesicle fusion, overcoming major drawbacks of conventional bulk fusion assays. The technique also allows us to track the dynamic transitions in a single fusion event with millisecond time resolution. With this powerful approach, we intend to reconstitute synchronized vesicle fusion in a test tube. Specifically, we test a mechanistic model proposing that Cpx arrests an intermediate stage called hemifusion and that Syt1 delivers the final blow to the fusion machinery at the Ca2+ spike. In parallel, this research program uses site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR), a powerful technique for the investigation of the protein structure at the membrane interface. With EPR we intend to comprehend the structural basis for the regulation of SNARE-dependent fusion necessary for synchronization. The combined approach of the single fusion assay and SDSL EPR will provide important insights into the mechanism by which the synchronized fast release is choreographed in the neuron.